Despite all the advances that have been achieved during the last 20 years, cancer is still one of the leading causes of mortality worldwide. Transitional cell bladder cancer is the most common cancer of the urinary tract; it is also the fourth most common cancer in men and the eight most common in women. Based on data from the International Agency for the Investigation of Cancer, GLOBOCAN, for the year 2000, more than 136.000 new cases per year are diagnosed in Europe, 13.000 in Japan and 56.000 in North America. More than 3-4 times this number of patients are treated and monitored at hospitals every year; and more than 49.000, 4.500 and 12.000 deaths are due to bladder cancer every year in Europe, Japan and North America, respectively (according to the International Agency for Research on Cancer GLOBOCAN 2000).
Transitional cell carcinoma (TCC) is the most common type of bladder cancer, accounting for more than 90% of all cases. The remaining cases are squamous cell carcinomas (7%), adenocarcinomas (2%), and undifferentiated carcinomas (1%).
Tumour grade and stage are the best prognostic indicators of transitional cell carcinoma of the bladder. Bladder tumours are graded cytomorphologically from G1 to G3 in decreasing state of differentiation and increasing aggressiveness of the disease according to the World Health Organization (WHO). With respect to stage or invasivity, TCCs of the bladder are classified as superficial papillary (Ta and T1), muscle invasive (T2 to T4), or the uncommon carcinoma in situ or tumour in situ (TIS).
Low-grade (G1) tumours are usually confined to the mucosa or infiltrate superficial layers (stage Ta and T1). Most high-grade tumours are detected at least at T1 stage (invading lamina propria). Approximately 75% of the diagnosed bladder cancer cases are superficial. The remaining 25% are muscle invasive at the moment of diagnosis.
The clinical importance of distinguishing superficial and invasive tumours stems from the need to perform radical cystectomy, with lymphadenectomy and bladder reconstruction in case of extended cancers (beyond the muscular layer). Tumours diagnosed in stages Ta and T1 allow the organ to be preserved and can be treated by transurethral resection and in some cases chemotherapy or intravesicular immunotherapy.
Patients with superficial TCC have a good prognosis but have a 70% risk of recurrence; these patients have to be monitored for tumour recurrence after treatment, following different protocols depending on the hospital, although the most frequent method is evaluation by the urologist every 3 months during the first 2 years, every 6 months for the following 2 years and every year thereafter. In spite of the high risk of recurrence, Ta tumours tend to be low grade and only 10-15% will progress to muscle invasion in 2 years; the percentage of T1 tumours that progresses to T2 stage is higher (30-50%).
Patients with invasive TCC have a poor prognosis; 50% of these patients at stage T2 or higher develop distant metastases within two years of diagnosis, and 69% of them die within 5 years. New diagnosis systems for early detection are needed given that 80-90% of patients with T2 or higher are first diagnosed at this highly aggressive stage and not in previous stages (de Vere White, R. W. and Stapp, E., Oncology, 1998, 12:1717-1723).
Currently, the best diagnostic system for bladder cancer in individuals presenting symptoms such as hematuria or dysuria, in the absence of infection, is cytoscopy. Based on statistical data of incidence and recurrence, it has been estimated that more than 500.000 cystoscopies are performed annually in the USA (van Rhijn, B. W. G., et al., Cancer Res., 2001, 61:1265-1268). Flexible cytoscopes are used to make the technique less aggressive, but it remains invasive and highly unpleasant, and it also requires some form of anaesthesia.
The prevailing non-invasive technique for diagnosis of transitional cell bladder cancer is to identify neoplastic cells by morphological examination of the cells in urine (Loh, C. S., et al., Br. J. Urol., 1996, 77:655-658). Cytology is currently used to follow up patients diagnosed with and treated for bladder cancer. On the other hand urine cytology can detect tumours in situ that are not detectable by cytoscopy as well as tumours located in the upper end of the bladder or the upper urinary tract, i.e. ureter, pelvis and renal, that are not easily accessible by endoscopy (Lotan, Y. and Roehrborn, J. Urol., 2002, 167:75-79).
Nevertheless several studies have shown that cytology has a very low sensitivity for bladder cancer diagnosis, missing up to 50% of tumours (Boman, H., et al., J. Urol., 2002, 167:80-83); in reality, there is no non-invasive method available to diagnose bladder cancer with high sensitivity and specificity (Boman, H., et al., J. Urol., 2002, 167:80-83). Such non-invasive methods would allow routine screening procedures for early detection of any transitional carcinoma including of the upper urinary tract, both de novo or in evaluating recurrence after treatment, including the detection of incipient invasive tumours or those at a high risk of developing aggressive disease.
Alteration of gene expression levels is tightly associated to uncontrolled cell growth and de-differentiation, common features of all cancers. The expression levels of the so-called “tumour suppressor genes”, which act to block malignant cell growth, are repressed in tumour cells; and expression levels of the so-called “oncogenes”, which act to induce malignant growth, are elevated in tumour cells.
Many of these genes have been associated to bladder cancer development, including Rb, p53, p16, p14ARF, cyclin D1 (Fujimoto, K., et al., Cancer Res., 1998, 52:1393-1398; Grossman, B. H., et al., Clin. Cancer Res., 1998, 8:829-834; Balazs, M., et al., Genes Chromosomes Cancer, 1997, 19:84-89). The alteration in the expression of these genes could be used as a diagnostic marker of transitional cell carcinoma of the bladder; among these proposed markers have been proposed nuclear matrix protein NMP22 (Soloway, M. S., et al., J. Urol., 1996, 156:363-367; Casella, R., et al., J. Urol, 2000, 164:1926-1928), Hyaluronic Acid and Hyaluronidase (Pham, H. T., et al., Cancer Res., 1997, 57:778-783; Hautmann, S. H., et al., J. Urol., 2001, 165:2068-2074), Basement Membrane Complexes (BTA) (Pode, D., et al., J. Urol., 1999, 161:443-446; Thomas, L., et al., Clin. Chem, 1999, 45:472-477, Carcinoembryonic antigen (CEA) (Halim, A. B., et al., Int. J. Biol. Markers, 1992; 7:234-239), Uroplakin II (Wu, X. R., et al., Cancer Res., 1998; 58:1291-1297), Scatter Factor/Hepatocyte Growth Factor (SF/HGF) (Gohji, K., et al., J. Clin. Oncol., 2000; 18:2963-2971), proteins of the keratin/cytokeratin family like cytokeratin 20 (Buchumensky, V., et al., J. Urol., 1998, 160:1971-1974), and cytokeratin 18 (Sánchez-Carbayo, M., et al., Clin. Cancer Res., 2000, 6:3585-3594), Mammary tumour 8-Ka Protein (MAT-8) (Morrison, B. W., et al., J. Biol. Chem., 1995, 270:2176-2182), Telomerase
However, it is likely that many of the genes involved in the initiation and progression of bladder cancer are currently unknown. No marker to predict the prognosis and extent of bladder cancer has been proven useful in clinical trials (Miyake et al., 2002). (Miyake, H., et al., J. Urol., 2002; 167:1282-1287). The identification of differentially expressed genes in bladder cell carcinoma could lead to the identification of biological markers, which could be of significant value for the diagnosis, prognosis and treatment of this disease.
Once transitional cell carcinoma of the bladder has been diagnosed, transurethral resection is carried out to treat superficial papillary tumours; superficial TIS and T1 are treated, in addition to transurethral resection, with intravesicular treatment with Bacillus-Calmette Guerin (BCG). If the cancer is muscle invasive, the patient is treated by radical cystectomy; if the patient will not tolerate this surgery, radiation therapy or chemotherapy is used. The 69% percent of the patients with muscle invasive TCC die within five years after diagnosis, even after receiving treatment. Alternative therapeutic approaches are necessary to treat muscle invasive TCC with a higher efficiency; also needed are alternative therapeutic approaches to treat low-grade tumours more efficiently than through surgery, or to complement surgery in order to avoid recurrences and progression of the tumour to an invasive state.
Fibroblast growth factors (FGF) are a family of more than twenty proteins involved in the regulation of biological processes including cell proliferation, cell differentiation, cell growth, cell migration, morphogenesis, angiogenesis and tissue remodelling. The FGFs bind with high affinity to cell surface receptors (Fibroblast Growth Factor Receptors, or FGFRs) that have tyrosine kinase activity. The protein kinases are a family of proteins, which effect the phosphorylation of other proteins and play a key role in the regulation of many cellular processes (Hanks, et al., Science 1988, 241, 42-52). When the FGF ligand binds to FGFR, the FGFR is converted to a dimeric active form that autophosphorylates in the kinase domain; then the activated FGFR binds and phosphorylates other effector proteins, thus starting a signal transduction pathway from the cell surface to the nucleus (Crews and Erikson. Cell. 1993. 74:215-217). The loss of regulation of growth factor signalling pathways is a frequent occurrence in cancer.
Four FGFRs have been identified to date: FGFR1 (also called Flg, fms-like gene, flt-2, bFGFR, N-bFGFR or Cek1), FGFR2 (also called Bek-Bacterial Expressed Kinase-, KGFR, Ksam, Ksaml and Cek3), FGFR3 (also called Cek2) and FGFR4. All mature FGFRs share a common structure consisting of an amino terminal signal peptide, three extracellular immunoglobulin-like domains (Ig domain I, Ig domain II, Ig domain III), with an acidic region between Ig domains I and II (the “acidic box” domain), a transmembrane domain, and intracellular kinase domains (Ullrich and Schlessinger, Cell 61:203, 1990; Johnson and Williams (1992) Adv. Cancer Res. 60:1-41). The distinct FGFR isoforms have different binding affinities for the different FGF ligands, thus FGF8 (androgen-induced growth factor) and FGF9 (glial activating factor) appear to have increased selectivity for FGFR3 (Chellaiah et al. J Biol Chem 1994; 269:11620).
Specific point mutations in FGFR3, that lead to the activation of its tyrosine kinase activity, have been previously associated to different syndromes related to bone development (Chen, H., et al. J. Clin. Invest., 1999, 104(11):1517-1525). Mutations in FGFR3 have also been detected in multiple myelomas (10-25% of tumours. Plowright et al. Blood 2000 Feb. 1; 95(3):992-8; Chesi et al. Blood 2001 Feb. 1; 97(3):729-36; Soverini et al. Haematologica 2002 October; 87(10):1036-40; Pollett et al. Blood 2002 Nov. 15; 100(10):3819-3821), in cervical carcinomas (3.5-25% of tumours. Sibley et al. Oncogene 2001 Jul. 19; 20(32):4416-8; Dai et al. Anal Cell Pathol 2001; 23(2):45-9) and in bladder carcinomas (Cappellen et al. Nat Genet 1999 September; 23(1):18-20; Sibley et al. Oncogene 2001 Feb. 8; 20(6):686-91; Sibley et al. Oncogene 2001 Jul. 19; 20(32):4416-8; Billerey et al. Am J Pathol. 2001 June; 158(6):1955-9) Activating FGFR3 mutations were detected in 40-50% of bladder tumours; the incidence was significantly higher, up to 80%, in low grade or superficial tumours than in high grade or invasive tumours; and the bladder cancer recurrence rates were clearly lower for tumours with a mutant FGFR3 (Kimura et al. Cancer 2001 Nov. 15; 92(10):2555-61; van Rhijn et al. Cancer Res 2001 Feb. 15; 61(4):1265-8).
Unexpectedly, the authors of the present invention have discovered, after thorough research and using different techniques, that the expression level of the FGFR3 gene and concentration of the protein is elevated in biopsies of bladder transitional cell carcinomas when compared with expression in normal bladder tissue and, moreover, the treatment of bladder cancer cell lines expressing high concentrations of FGFR3 with antibody against FGFR3 protein produce inhibition of cell proliferation of bladder cancer cell lines.
The authors of the present invention have also surprisingly discovered that the elevated levels of FGFR3 protein expression are predominantly associated with superficial tumours.
The present invention, therefore, provides a highly sensitive in vitro method to detect the presence of a bladder carcinoma, to determine the stage or severity of this cancer in an individual or to monitor the effect of therapy administered to an individual with the said cancer. Also, the present invention provides targets or tools for the screening, identification, development and evaluation of the efficacy of therapeutic compounds for the treatment of cancer of the bladder, particularly for tumour treatment, as neoadjuvant before resection or as adjuvant after resection with the aim of reducing recurrence and progression. Finally, the invention provides agents characterised by the fact that they inhibit expression and/or activity of the FGFR3 protein for the treatment of cancer of the bladder.